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Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations


Researchers have several options when designing proteomics experiments. Primary among these are choices of experimental method, instrumentation and spectral interpretation software. To evaluate these choices on a proteome scale, we compared triplicate measurements of the yeast proteome by liquid chromatography tandem mass spectrometry (LC-MS/MS) using linear ion trap (LTQ) and hybrid quadrupole time-of-flight (QqTOF; QSTAR) mass spectrometers. Acquired MS/MS spectra were interpreted with Mascot and SEQUEST algorithms with and without the requirement that all returned peptides be tryptic. Using a composite target decoy database strategy, we selected scoring criteria yielding 1% estimated false positive identifications at maximum sensitivity for all data sets, allowing reasonable comparisons between them. These comparisons indicate that Mascot and SEQUEST yield similar results for LTQ-acquired spectra but less so for QSTAR spectra. Furthermore, low reproducibility between replicate data acquisitions made on one or both instrument platforms can be exploited to increase sensitivity and confidence in large-scale protein identifications.

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Figure 1: Overview of experiment design.
Figure 2: Mascot and SEQUEST are complementary analysis tools for interpreting LTQ and QSTAR MS/MS.
Figure 3: Considerably more peptide and protein identifications can be achieved by analyzing a sample multiple times.
Figure 4: LTQ and QSTAR mass spectrometers exclusively identify many peptides and proteins.
Figure 5: Practical comparison of analytical options.


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This work was supported in part by US National Institutes of Health GM67945 and HG00041 (S.P.G.). We thank D. Moazed for yeast lysate and the Pathology Functional Proteomic Center at Harvard Medical School for allowing use of their Mascot server.

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Correspondence to Steven P Gygi.

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Competing interests

ThermoElectron holds the licensing rights for the distribution of the SEQUEST algorithm. The Gygi lab has an industry-sponsored research project with ThermoElectron. These funds, however, did not provide any support (materials, equipment or salary) for the research described in this manuscript.

Supplementary information

Supplementary Fig. 1

Mascot and SEQUEST score distributions for target (blue) and decoy (red) database hits. (PDF 3286 kb)

Supplementary Fig. 2

Examples of MS/MS acquired by LTQ or QSTAR mass spectrometers. (PDF 107 kb)

Supplementary Fig. 3

Representation of MS/MS quality on LTQ and QSTAR and its influence on Mascot and SEQUEST scoring. (PDF 913 kb)

Supplementary Fig. 4

Comparison of LTQ and QSTAR acquisitions from equal gradient analyses. (PDF 73 kb)

Supplementary Fig. 5

Proteins identified by just one instrument appear to be less abundant than those identified by both LTQ and QSTAR. (PDF 83 kb)

Supplementary Fig. 6

Differential preference for ions selected for MS/MS by LTQ and QSTAR mass spectrometers influences the length distribution of identified peptides. (PDF 338 kb)

Supplementary Table 1

Mascot and SEQUEST score filter criteria applied to MS/MS spectra acquired on the LTQ and QSTAR mass spectrometers to achieve ˜99% precision (1% false positive rate) at maximum estimated sensitivity. (PDF 61 kb)

Supplementary Methods (PDF 83 kb)

Supplementary Data (PDF 87 kb)

Supplementary Discussion (PDF 109 kb)

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Elias, J., Haas, W., Faherty, B. et al. Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations. Nat Methods 2, 667–675 (2005).

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